In human development, the genesis and further differentiation of retinal tissue follows a well-defined and conserved developmental program, with numerous markers available to distinguish the major stages of retinogenesis. Retinogenesis begins within the first few weeks of human development, when a portion of the primitive anterior neuroepithelium gives rise to the paired eye fields (Li, H., et al., 1997; Mathers, P. H., et al., 2000; Bailey, T. J., et al., 2004; Zuber, M. E., et al., 2003). The eye fields are made up of a cell population characterized by the expression of numerous transcription factors, including Pax6, Rx, Otx2, Six3, Six6, TII and Lhx2. Although Pax6 and Rx have been used to identify retinal progenitor cells (RPC) in differentiating embryonic stem cell (ESC) cultures (Osakada, F., et al., 2008; Mathers, P. H., et al., 2000), during development Pax6 and Rx are initially co-expressed in a broad region of the anterior neural plate that includes the eye field and future forebrain (Mathers, P. H., et al., 2000). Thereafter, Pax6+/Rx+ cells become restricted to more specific areas of the developing CNS (Mathers, P. H., et al., 2000), predominantly the retina (Bailey, T. J., et al., 2004; Furukawa, T., et al., 1997b). The remaining cells predominantly develop into forebrain structures.
The next in vivo retinal specification phase involves formation of optic vesicles from the paired eye fields. After the optic vesicles evaginate from the paired eye fields, all cells that will give rise to either the neural retina or the retinal pigment epithelium (RPE) express the transcription factor Mitf (Chow, R. L., et al., 2001; Bharti, K., et al., 2008).
The subset of Mitf+ cells destined to become neural retina or retinal pigment epithelium subsequently downregulate Mitf in response to the onset of expression of Chx10, also called Vsx2 (Horsford, D. J., et al., 2005; Rowan, S., et al., 2004). Neural retinal progenitors destined for the inner layer of the optic cup express Chx10 and downregulate Mitf in response to fibroblast growth factors (FGFs) secreted by the overlying surface ectoderm. Thus, Chx10 is the earliest specific marker of neural RPC within the optic vesicle and cup (Rowan, S., et al., 2004). Chx10+ retinal progenitors give rise to all cell types of the neural retina: cones, rods, ganglion cells, amacrine cells, bipolar cells, horizontal cells and Muller glia. Conversely, cells destined for the outer layer of the optic cup remain Mitf+ and Chx10-negative and subsequently differentiate into RPE.
Among the first differentiated neural retinal phenotypes observed during development are cone photoreceptors (Barishak, Y., 2001; Finlay, B. L., 2008), whose precursors express the primitive cone and rod photoreceptor-specific transcription factor Crx (Chen, S., et al., 1997; Furukawa, T., et al., 1997). Later, cones express recoverin and ultimately opsin. Rod photoreceptors express the transcription factor Nrl followed by the phototransduction molecules recoverin and rhodopsin. Retinal ganglion cells are also produced early on, and can be distinguished among developing retinal cells by their expression of βIII tubulin and HuC/D and by their long processes. Other retinal neurons such as bipolar cells, horizontal cells and amacrine cells have markers as well (PKCα, calbindin and calretinin, respectively). Again, however, these markers are found elsewhere in the central nervous system, so it is imperative that the population from which they arise be established as neural retinal progenitors (Chx10+/Pax6+), which themselves come from optic vesicle and eye field cells.
Retinal development is of particular interest to clinicians and researchers, because millions of individuals in North America suffer varying degrees of irreversible vision loss as a result of retinal degenerative disease (RDD). Inherited and acquired outer RDDs, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD), are major causes of progressive vision loss for which there are no cures and few therapeutic options. In such disorders, rod and cone photoreceptor cells and adjacent retinal pigment epithelium (RPE) cells in the outer retina are most affected. Inner RDDs predominantly affect retinal ganglion cells, causing glaucoma and other diseases that result in permanent vision loss. During the early and middle stages of RDD, treatment focuses on rescuing at-risk cells and preserving visual function. After an RDD results in a critical level of cell death, suitable treatment approaches are limited to bypassing or replacing lost cells while mitigating the underlying disease process.
Because neural tissue is generally not self-regenerating, successfully treating any neurodegenerative disease is difficult. However, because the outer retina is easily accessible and contains a comparatively simple network of short-range intercellular connections, the outer retina is a more favorable treatment target than most other central nervous system tissue.
MacLaren et al. (Nature, 2006, 444:203-207) demonstrated therapeutic replacement of outer retinal cells in a mouse RP model by showing that rod precursor allografts could integrate and restore partial retinal function. McLaren's proof of concept spurred efforts to find comparably capable sources of human cells having the potential to expand in culture and differentiate into multiple retinal cell types. However, cells from proposed sources often have characteristics that significantly limit potential clinical use. For example, human fetal retinal progenitor cells (RPC) have been propagated in culture, but over time, the cells became progressively restricted to a glial fate, necessitating gene misexpression to generate neuronal cell types (Gamm et al, Stem Cells 2008). Similarly, RPE, iris pigment epithelium and non-ocular stem and progenitor cells often lack a definitive capacity to produce retinal cells, and the existence of a multipotent retinal stem cell population in adult human pigmented ciliary epithelium was recently called into question (Cicero et al., 2009; Gualdoni et al, 2010).
Although human fetal forebrain progenitors have proven to be effective for reducing anatomical and functional photoreceptor loss and visual decline after subretinal transplantation in rodent models of RDD (likely due to their ability to secrete natural neuroprotective factors), finding human sources that work for retinal cell replacement has been problematic.
The successful culturing of human pluripotent stem cells, including both embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC), has provided an intriguing and potentially inexhaustible supply of cells with regenerative potential. Additionally, human ESC and iPSC have potential as research tools for studying the developmental steps leading to the production of retinal cell types, the most important being photoreceptors (cones and rods), ganglion cells and RPE. More detailed knowledge of the steps involved in the differentiation of these and other retinal cell types would be useful for both basic science and clinical studies, as it would improve cell production efficiency, reproducibility, and perhaps also cell function.
Furthermore, if they can be used to provide model systems that successfully replicate human retinogenesis in vivo, human ESC and iPSC cells could potentially provide a powerful tool for examining early human retinal and neural cell development at stages that were previously inaccessible. One criterion for assessing pluripotent stem cell-based developmental model systems is the capacity to recapitulate the normal embryonic maturation sequence in a controlled, stepwise fashion (Keller, G., 2005; Pera, M. F., et al., 2004). Such systems should also provide the opportunity to test the effects of developmental stimuli and enrich for early cell populations to reduce contamination from undesired and/or unidentified cell lineages. It would also be advantageous to monitor cellular maturation by marker expression to ascertain whether developmental checkpoints are met in order and according to a predictable timeline.
Human iPSC are a subclass of human pluripotent cells created by reprogramming somatic cells such as skin fibroblasts or other mature cell types to a pluripotent state by transiently misexpressing a few select genes (Takahashi, K., et al., 2007; Yu, J., et al., 2007). Early studies indicate that human iPSC can have widely varying innate potential to produce neuroepithelial cells, the predecessors of all retinal cell types (Hu et al., 2010, Yu et al., 2007; Hirami et al., 2009). Because the differentiated cells derived from iPSC are genetically identical to the adult cells from which the iPSC are derived, iPSC have a potential advantage over ESC in certain therapeutic or research applications. For example, IPSC technology offers an alternative to human ESC differentiation wherein it is envisioned that one can produce iPSC from somatic cells of an individual and then treat the same individual with cells (e.g., retinal lineage cells) obtained by differentiating the iPSC. In addition, individual-specific pluripotent iPSC lines can be used to develop in vitro models of human diseases. (Ebert, A. D., et al, 2009; Park, I. H., et al, 2008).
The therapeutic and research potential of human ESC and human iPSC would be enhanced if the earliest committed cells in the retinal lineage could be isolated from unwanted or contaminating cell types into a substantially pure cell culture. This is particularly the case for retinal neurons, which, with the exception of photoreceptors, cannot be unequivocally identified unless one is sure that they were derived from retinal progenitor cells. Similarly, the study and use of human ESC- and iPSC-derived forebrain cells would be aided by a method that produces enriched populations of these cells at a very early stage of differentiation.
Current methods for differentiating pluripotent cells into cell types of interest have limited clinical and scientific appeal due to contamination from early, unwanted cell types and a lack of information regarding the key steps involved in genesis of the differentiated cells. Existing methods have focused on deriving mixed retinal cell populations or more mature cells such as RPE (U.S. Pat. Nos. 7,541,186 and 7,736,896; Klimanskaya, I., et al., 2004; Vugler, A, et al., 2008; Clegg et al., 2009) or photoreceptors (Osakada, F., et al., 2008) using various exogenous factors to increase the percentage of early retinal cell types in the heterogeneous population of differentiating human ESC. For example, retinoic acid and taurine can induce human ESC to differentiate to photoreceptor-like cells (Osakada, F., et al., 2008). However, no one has described a method for differentiating human pluripotent cells into a highly enriched, isolated population of early retinal progenitor cells (RPC) that can progress through the major retinal developmental stages leading to production of mature cell types. Furthermore, retinal cell types produced thus far have not exhibited a differentiation time course comparable to that observed in normal human retinogenesis. Indeed, the timing of appearance in culture of selected retinal development stages has varied widely among published protocols (Banin, E., et al., 2006; Lamba, D A., et al., 2006; Osakada, F., et al., 2008; Klassen, H., et al., 2008). For example, the reported onset of expression of the Crx marker has ranged from one to thirteen weeks, depending on the protocol used (Lamba, D A., et al., 2006; Osakada, F., et al., 2008).
Thus, there is a need in the art for substantially pure cultures of certain human neuroepithelial lineage cells, including retinal progenitor cells, forebrain progenitor cells, and retinal pigment epithelium cells, that accurately model in vitro differentiation and development, and for simplified methods of producing such cultures.